Not applicable.
Not Applicable.
(1) Field of the Invention
The present invention relates to a human gene sequence that encodes the CDDF-1 polypeptide, which is involved in cell differentiation, in particular differentiation of undifferentiated or immature cells into neutrophils, monocytes/macrophages, mast cells and/or erythroid cells. The present invention further relates to assays for detecting CDDP-1 expression, vectors that express CDDP-1, methods for using CDDP-1 to induce or promote cell differentiation, and transgenic animals deficient in one or both CDDP-1 alleles. The present invention further relates to methods of gene therapy which comprise the administration of the above vectors to a human or animal.
(2) Description of Related Art
Development in higher eukaryotes proceeds by the differential, temporal, and tissue specific expression of particular genes. Among the factors which control gene expression are positive transcription factors that increase gene expression by binding to specific DNA sequences called enhancer sequences. These factors are called enhancer binding proteins. One such family of enhancer binding proteins is the CCAAT/enhancer binding protein (C/EBP) family.
The C/EBP family of transcription factors has been implicated in both the regulation of differentiation and differentiated function in myelomonocytic cells. C/EBPxcex1, C/EBPxcex2, and C/EBPxcex4 are temporally regulated in the granulocyte-colony stimulating factor (G-CSF)-stimulated differentiation of 32D clone 3 myeloblasts into granulocytes (Scott et al., Blood 80: 1725-1735 (1992)). C/EBPxcex1 is expressed at high levels early in differentiation and decreases to an undetectable level in terminally differentiated cells, while C/EBPxcex2 and C/EBPxcex4 are upregulated as the cells differentiate.
Similarly, C/EBPxcex2 is upregulated in macrophage differentiation (Natsuka et al., Blood 79: 460-466 (1992)). However, observations in primary cells and in bipotential precursor cell lines yield a somewhat different pattern of C/EBPxcex1 expression (Radomska et al., Mol. Cell. Biol. 18: 4301-4314 (1998)). In these cell lines, C/EBPxcex1 is first expressed when multipotential cells become committed to the myeloid lineage, then C/EBPxcex1 expression is upregulated and its expression maintained during granulocytic expression, then its expression is rapidly down regulated during monocytic differentiation. Consistent with its early appearance and high level of expression in granulocyte differentiation, conditional expression of C/EBPxcex1 in transfected bipotential cells induces neutrophilic differentiation and blocks monocytic differentiation (Radomska et al. ibid.). C/EBPxcex1 has been shown to induce expression of the G-CSF receptor and the interleuken-6 (IL-6) receptor. Both receptors are involved in cell development.
Genetically created C/EBPxcex1-deficient mice are completely blocked in the development of neutrophils and eosinophils (Zhang et al., Proc. Natl. Acad. Sci. USA 94: 569 (1997)). While these mice no longer express the C/EBPxcex1-regulated G-CSF receptor, lack of G-CSF expression alone is not responsible for the blocked development because mice deficient in G-CSF receptor expression are still capable of producing mature neutrophils, albeit in numbers that are reduced compared to that in normal mice (Liu et al., Immunity 5: 491-501 (1996)). The IL-6 receptor is either directly or indirectly regulated by C/EBPxcex1, and IL-6 signaling is important for granulopoiesis; however, the genetically created IL-6 receptor and the G-CSF receptor deficient mice are similar to mice made deficient for only the G-CSF receptor (Zhang et al., J. Exp. Med. 188: 1173-1184 (1998)). Therefore, there must be other important C/EBPxcex1 genes other than those encoding the G-CSF receptor and the IL-6 receptor.
Consistent with peak expression late in myeloid differentiation, C/EBPxcex2 and C/EBPxcex4 are effectors in the induction of proinflammatory genes responsible for LPS, IL-1, or IL-6 stimulation (Akira et al., EMBO J. 9: 1897-1906 (1990); Kinoshita et al., Proc. Natl. Acad. Sci. USA 89: 1473-1476 (1992)). The DNA binding activity of C/EBPxcex2 is increased by a post-transcriptional mechanism when cells are exposed to LPS, IL-1, or IL-6 (Akira et al. ibid.; Poli et al., Cell 63: 643-653 (1990)), and C/EBPxcex2 and C/EBPxcex4 mRNA levels can be induced by LPS, IL-1, or IL-6 (Akira et al. ibid.; Kinoshita et al. ibid.). C/EBPxcex2 expression is capable of enhancing transcription from promoters containing elements responsive to those factors (Akira et al. ibid.; Poli et al. ibid.; Chang et al., Mol. Cell. Biol. 10: 6642-6653 (1990); Descombes et al., Genes Dev. 4: 1541-1551 (1990)). Both C/EBPxcex2 and C/EBPxcex4 can transactivate a reporter gene regulated by the IL-6 promoter in transient assays (Akira et al. ibid.; Kinoshita et al. ibid.). Furthermore, the promoter regions of the genes for IL-6, IL-1xcex1, IL-1xcex2, IL-8, tumor necrosis factor alpha (TNFxcex2), G-CSF, macrophage inducible nitric oxide synthase (iNOS), and lysozyme (Akira et al. ibid.; Furutani et al., Nuc. Acids Res. 14: 3167-3179 (1986); Lowenstein et al., Proc. Natl. Acad. Sci. USA 90: 9730-9734 (1993); Natsuka et al., Blood 79: 460-466 (1992); Shirakawa et al., Mol. Cell. Biol. 13: 1332-1344 (1993); Zhang and Rom, Mol. Cell. Biol. 13: 3831-3837 (1993)) all contain C/EBP binding motifs. Furthermore, ectopic expression of C/EBPxcex1, C/EBPxcex2, or C/EBPxcex4 confers LPS-inducibility of IL-6 and MCP-1 expression to cell lines that normally lack C/EBP expression (Bretz et al., Proc. Natl. Acad. Sci. USA 91: 7306-7310 (1994); Hu et al., J. Immunol. 160: 2334-2342 (1998)).
C/EBPxcex5 is unique among the transcription factors of the C/EBP family because it is myeloid-specific in its expression (Williams et al., J. Biol. Chem. 273: 13493-13501 (1998); Chumakov et al., Mol. Cell. Biol. 17: 1375-1386 (1997)). In particular, C/EBPxcex5 is expressed in maturing neutrophils (Chih et al., Blood 90: 2987-2994 (1997); Morosetti et al., Blood 90: 2591-2600 (1997); Yamanaka et al., Proc. Natl. Acad. Sci. USA 94: 6462-6467 (1997)). C/EBPxcex5-deficient mice fail to develop functional neutrophils and eosinophils (Yamanaka et al., Proc. Natl. Acad. Sci. USA 94: 13187-13192 (1997)) with a block later in the differentiation process than that seen in C/EBPxcex1-deficient mice. Consistent with a block later in the differentiation process, C/EBPxcex5 has been found to transactivate, in cooperation with c-myb, the promoters for mim-1 and neutrophil elastase in transient expression assays (Verbeek et al., Blood 93: 3327-3337 (1999)), and to upregulate endogenous expression of a number of genes encoding proinflammatory cytokines including IL-6, MCP-1, MIP-1xcex1, and MIP-1xcex2 (Williams et al. ibid.).
C/EBP family members have also been implicated in the differentiation of the erythroid and B cell lineages. Erythropoietin upregulates expression of CHOP, a C/EBP family member that acts as a dominant negative regulator of other C/EBP family members (Coutts et al., Blood 93: 3369-3378 (1999)). Overexpression of CHOP in Rauscher cells enhanced erythropoietin and DMSO-induced differentiation, while inhibition of CHOP expression reduced CFU-E formation. C/EBPxcex2 is also upregulated in the course of erythroid differentiation. Mice deficient in C/EBPxcex2 expression are impaired in the generation of bone marrow B lymphocytes (Chen et al., Blood 90: 156-164 (1997)). These mice showed decreased expression of IL-7 by bone marrow stromal cells as well as defective IL-7 signaling. C/EBPxcex2 has also been shown to be a component of a DNA binding complex that activates the Id1 gene, a negative regulator of differentiation in pro-B cells (Saisanit and Sun, Mol. Cell. Biol. 17: 844-850 (1997)). This suggests that C/EBPxcex2 participates in blocking differentiation and promoting proliferation at the pro-B cell stage, but promotes differentiation through the IL-7 pathway at later stages of differentiation.
Thus, the C/EBP family of transcription factors are important in hematopoietic differentiation and have a particularly central role in myelomonocytic differentiation. Since the currently identified target genes of C/EBP regulation seem inadequate to fully explain the role of these transcription factors in myelomonocytic differentiation, there is a need to identify other genes which have an important role in differentiation. These genes would provide tools which could be used in gene therapy or other therapeutic treatments to induce differentiation in abnormal cells that are undifferentiated such as those cells found in leukemias.
The present invention provides a human gene sequence that encodes the CDDP-1 polypeptide, which is involved in cell differentiation, in particular differentiation of undifferentiated or immature cells into neutrophils, monocytes/macrophages, mast cells and/or erythroid cells. The present invention further provides assays for detecting CDDP-1 expression, vectors that express CDDP-1, methods for using CDDP-1 to induce or promote cell differentiation, and transgenic animals deficient in one or both CDDP-1 alleles. The present invention further pertains to methods of gene therapy that comprise the administration of vectors expressing CDDP-1 to a human or animal, and for treatment of diseases that are a result of proliferation of undifferentiated cells including, but not limited to myeloid progenitor cells.
Thus, the present invention provides an isolated DNA molecule that encodes human C/EBP Differential Display Product-1 (CDDP-1) or mutant thereof wherein the CDDP-1 promotes differentiation of undifferentiated cells along a pathway of differentiation. In particular, an isolated DNA molecule that encodes human C/EBP Differential Display Product-1 (CDDP-1) or mutant thereof wherein the CDDP-1 promotes differentiation of myeloid progenitor cells along a neutrophilic, monocytic, or macrophage differentiation pathway; or an isolated DNA molecule that encodes human C/EBP Differential Display Product-1 (CDDP-1) or mutant thereof wherein the CDDP-1 promotes differentiation of hematopoietic cells along an erythroid or mast cell differentiation pathway. Most particularly, an isolated DNA molecule that encodes human C/EBP Differential Display Product-1 (CDDP-1) or mutant thereof wherein the CDDP-1, which is localized to the cytoplasm, promotes differentiation of myeloid progenitor cells into cells selected from the group consisting neutrophils, monocytes, macrophages, and combinations thereof wherein induction of CDDP-1 in neutrophilic differentiation is biphasic occurring with myeloperoxidase induction and then again with lactoferrin induction and expression of CDDP-1 in monocyte differentiation displays a pattern of reduced expression. In particular, the isolated DNA molecule wherein the expressed CDDP-1 comprises the amino acid sequence represented by SEQ ID NO:2 or mutant thereof, or wherein the DNA molecule encoding CDDP-1 comprises the nucleic acid sequence represented by SEQ ID NO:1 or mutant thereof. Thus, in a preferred embodiment, the present invention provides an isolated human DNA encoding CDDP-1 comprising the nucleotide sequence set forth in SEQ ID NO:1 or mutant thereof. In a further preferred embodiment, the DNA molecule is detectably labeled. Further still, the present invention provides an isolated CDDP-1 protein comprising the amino acid sequence set forth in SEQ ID NO:2.
The present invention also provides a vector, preferably a vector comprising a retrovirus or plasmid, comprising a DNA molecule that encodes human C/EBP Differential Display Product-1 (CDDP-1) or mutant thereof wherein the CDDP-1 promotes differentiation of undifferentiated cells along a pathway of differentiation. In particular, a vector comprising a DNA molecule that encodes human C/EBP Differential Display Product-1 (CDDP-1) or mutant thereof wherein the CDDP-1 promotes differentiation of myeloid progenitor cells along a neutrophilic, monocytic, or macrophage differentiation pathway; a vector comprising a DNA molecule that encodes human C/EBP Differential Display Product-1 (CDDP-1) or mutant thereof wherein the CDDP-1 promotes differentiation of hematopoietic cells along an erythroid or mast cell differentiation pathway; or, a DNA molecule encoding a human C/EBP Differential Display Product-1 (CDDP-1) or mutant thereof, which is involved in differentiation of myeloid progenitor cells into cells selected from the group consisting of neutrophils, monocyte, macrophages and combinations thereof. In particular, the vector wherein the encoded CDDP-1 comprises the amino acid sequence of SEQ ID NO:2 or mutant thereof or wherein the CDDP-1 is encoded by the DNA molecule comprising the nucleic acid sequence of SEQ ID NO:1 or mutant thereof. Preferably, the present invention provides a vector wherein the CDDP-1 is capable of inducing differentiation of mammalian cells. Particularly, wherein the mammalian cells comprises myeloid progenitor cells. In a preferred embodiment, the vector contains DNA comprising the nucleotide sequence set forth in SEQ ID NO:1 or mutant thereof.
The present invention also provides a cell line harboring a vector containing DNA comprising the nucleotide sequence set forth in SEQ ID NO:1 or mutant thereof. The present invention also provides a transgenic animal which is heterozygous or homozygous CDDP-1 deficient. Preferably, the transgenic animal is a mouse.
The present invention further provides a method for determining whether cells express mRNA encoding C/EBP Differential Display Product-1 (CDDP-1), which comprises (a) providing a detectably labeled nucleic acid probe complementary to the mRNA encoding CDDP-1, (b) isolating the mRNA from the cells, (c) hybridizing the probe to the isolated RNA, and (d) detecting the probe hybridized to the mRNA. In particular, the method wherein the cells comprise myeloid progenitor cells. Preferably, the probe comprises the nucleotide sequence represented in SEQ ID NO.1 or mutant thereof.
The present invention further still provides a method for determining whether cells express C/EBP Differential Display Product-1 (CDDP-1), which comprises (a) providing a detectably labeled antibody that binds the CDDP-1, (b) isolating proteins from the cells, (c) reacting the proteins with the antibody, and (d) detecting the antibody bound to the CDDP-1. In particular, the method wherein the cells comprise myeloid progenitor cells. Preferably, the CDDP-1 comprises the amino acid sequence represented in SEQ ID NO.2 or mutant thereof.
In a method further still, the present invention provides a method for regulating differentiation of cells, which comprises (a) transfecting a vector containing an isolated DNA comprising the nucleotide sequence set forth in SEQ ID NO:1 or mutant thereof encoding CDDP-1 in the cells, and (b) inducing the cells to produce the CDDP-1 to regulate the differentiation of the cells. In particular, a method wherein the cells which produce CDDP-1 are in a mammal and wherein the cells are cultured in vitro.
Further still, the present invention provides a method for testing a chemical agent to determine its effect on cells that express CDDP-1, which comprises (a) culturing cells transfected with a vector containing DNA encoding CDDP-1 as set forth in SEQ ID NO:1 or mutant thereof encoding CDDP-1, (b) introducing the chemical agent into the culture of the cells, and (c) determining the affect of the chemical agent on the cells. In particular, wherein the effect on the cells that is determined, is differentiation of the cells. Preferably, the cells are myeloblast cells. In particular, the method wherein the cells are myeloid leukemic cells or progenitor myeloid progenitor cells. In one embodiment, the chemical agent is retinoic acid. Preferably, in determining the effect, the method includes control cells that do not express CDDP-1.
Further still, the present invention provides a method for inducing or promoting differentiation of undifferentiated cells in a mammal, which comprises introducing a vector containing DNA encoding CDDP-1 protein into the undifferentiated cells of the mammal to induce or promote differentiation of the undifferentiated cells. Preferably, a method for inducing cell differentiation in a mammal, which comprises introducing a vector containing DNA comprising the nucleotide sequence set forth in SEQ ID NO:1 or mutant thereof encoding CDDP-1 into the cells of the mammal. In a particular embodiment, a chemical agent is introduced into the animal with the vector to further augment the effects produced through the production of CDDP-1 encoded by the DNA, preferably the chemical agent is retinoic acid. Preferably, the CDDP-1 comprises the amino acid sequence set forth in SEQ ID NO:2. In a preferred embodiment, the undifferentiated cells are myeloid cells.
Further still, the present invention provides a kit for determining whether cells express C/EBP Differential Display Product-1 (CDDP-1). The kit comprises (a) providing a detectably labeled antibody that binds the CDDP-1, and (b) a means for determining the amount of the antibody bound to the CDDP-1. In particular, wherein the cells comprise myeloid progenitor cells and wherein the CDDP-1 comprises the amino acid sequence represented in SEQ ID NO.2 or mutant thereof. Alternatively, the present invention provides a kit for determining the level of CDDP-1 produced by a cell, which comprises (a) a protein binding reagent, which forms a complex with the CDDP-1, and (b) means for determining the level of the reagent or CDDP-1 in the complex. In particular wherein the cells are leukemic.
Further still, the present invention provides a kit for determining whether cells express RNA encoding C/EBP Differential Display Product-1 (CDDP-1). The kit comprises (a) providing a detectably labeled nucleic acid probe that hybridizes the RNA encoding CDDP-1, and (b) a means for detecting the probe hybridized to the RNA encoding CDDP-1. In particular, wherein the cells comprise myeloid cells and wherein the nucleic acid probe comprises the nucleic acid sequence represented in SEQ ID NO.1 or mutant thereof.
Finally, the present invention provides a transgenic animal wherein the transgenic animal is heterozygous or homozygous CDDP-1 deficient. Preferably, the transgenic animal is a transgenic mouse that is deficient for one or both CDDP-1 alleles.
An object of the present invention is to provide a polypeptide and a nucleic acid encoding the polypeptide wherein the polypeptide is involved in the differentiation of myeloid progenitor cells, in particular leukemia cells.
Another object of the present invention is to provide a method for identifying chemical agents that effect differentiation of myeloid cells by inducing expression of CDDP-1.
A further object of the present invention is to provide a method for inducing differentiation of leukemia cells in a patient.